The bio-processing industry has traditionally used stainless steel systems and piping in manufacturing processes for fermentation and cell cultivation. These devices are designed to be steam sterilized and reused. Cleaning and sterilization are however costly labour-intensive operations. Moreover, the installed cost of these traditional systems with the requisite piping and utilities is often prohibitive. Furthermore, these systems are typically designed for a specific process, and cannot be easily reconfigured for new applications. These limitations have led to adoption of a new approach over the last ten years—that of using plastic, single-use disposable bags and tubing, to replace the usual stainless steel tanks.
In particular bioreactors, traditionally made of stainless steel, have been replaced in many applications by disposable bags which are rocked to provide the necessary aeration and mixing necessary for cell culture. These single-use bags are typically provided sterile and eliminate the costly and time-consuming steps of cleaning and sterilization. The bags are designed to maintain a sterile environment during operation thereby minimizing the risk of contamination.
One of the successful disposable bioreactor systems uses a rocking table on to which a bioreactor bag is placed. The bioreactor bag is partially filled with liquid nutrient media and the desired cells. The table rocks the bag providing constant movement of the cells in the bag and also efficient gas exchange from the turbulent air-liquid surface. The bag, typically, has at least one gas supply tube for the introduction of air, carbon dioxide, nitrogen or oxygen, and at least one exhaust gas tube to allow for the removal of respired gases. Nutrients can be added through other tubes.
During cultivation, the cells produce metabolites, e.g. ammonium ions and lactate, which have an inhibitory effect on cells. This effect becomes an issue particularly in cultivation at high cell densities, which are required for cost-effective production of biopharmaceuticals such as therapeutic proteins or virus antigens. One way to reduce the concentrations of inhibitory metabolites is to use perfusion cultivation where culture medium is bled off by hydraulic flow through a filter which retains the cells but lets the metabolites and proteins pass through the filter. Expressed proteins can then be recovered from the filtrate and fresh culture medium is continuously supplied to the bioreactor to compensate for the lost liquid.
Due to the hydraulic flow through the filter, perfusion culture at high cell densities has issues with fouling and clogging of the filters. It is also in many cases desirable to retain the expressed protein in the bioreactor for recovery in a harvest step after cultivation. An attractive solution to these issues is to use dialysis cultivation, as described e.g. in R Poertner et al. Appl Microbiol Biotechnol (1998) 50: 403-414. Here, the cell culture is in contact with a dialysis membrane having a cut-off value chosen such that low molecular metabolites diffuse through the membrane, while cells and proteins are retained. There is no significant pressure drop over the membrane, meaning that mass transport is primarily by diffusion and little clogging or fouling will occur.
Dialysis cultivation is most commonly carried out with an external dialysis module, through which the culture is conveyed in a circuit outside the bioreactor. This is inconvenient as it increases the risks of a) contaminating the culture, b) leakage or catastrophic loss of the potentially biohazardous culture and c) attrition of sensitive cells by pumping through the circuit. Internal dialysis modules, placed inside the bioreactor have been described in e.g. Poertner, U.S. Pat. Nos. 5,576,211 and 6,933,144, but they are designed for rigid wall bioreactors such as stainless steel reactors or rolling bottles and the arrangements are not suitable for flexible bag bioreactors. They also tend to give low mass transport rates, which is detrimental to the process efficiency.
Accordingly there is a need for flexible bag bioreactors with internal dialysis modules suitable for dialysis cultivation with high mass transport rates.